The present invention relates to glycosyltransferase enzymes and the genes corresponding to such enzymes. In particular, the present invention relates to the enzyme N-acetylgalactosaminyltransferase. Specifically, the invention relates to the isolation and sequencing of the enzyme N-acetylgalactosaminyltransferase. The invention also relates to the construction of proteins capable of expressing the acceptor peptide for the enzyme N-acetylgalactosaminyltransferase.
Carbohydrates are an important class of biological compounds. In cells, carbohydrates function as structural components where they regulate viscosity, store energy, or are key components of cell surfaces. Nearly all site specific intercellular interactions involve cell surface carbohydrates. For example, union of sperm and egg as well as the implantation of fertilized egg are both mediated by cell surface carbohydrates. Likewise, a number of proteins that function as cell adhesion molecules, including GMP-140, ELAM-1, and lymphocyte adhesion molecules like Mel-14, exhibit structural features that mimic lectins, and are thought to bind specific cell surface carbohydrate structures (Stoolman, Cell (1989) 56:907-910). Glycosylated proteins as tumor-associated antigens are now being used to identify the presence of numerous carcinomas. Even isolated oligosaccharides have been found to exhibit biological activity on their own.
Specific galactose glycosaccharides are known to inhibit the agglutination of uropathogenic coliform bacteria with red blood cells (U.S. Pat. No. 4,521,592). Other oligosaccharides have been shown to possess potent antithrombic activity by increasing the levels of plasminogen activator (U.S. Pat. No. 4,801,583). This same biological activity has been used, by binding oligosaccharides, in conjunction with an amino glycoprotein, to medical instruments to provide medical surfaces which have anticoagulation effects (U.S. Pat. No. 4,810,784). Still other oligosaccharides have found utility as gram positive antibiotics and disinfectants (U.S. Pat. Nos. 4,851,338 and 4,665,060). Further, oligosaccharides have been used as bacteria receptor sites in the diagnosis and identification of specific bacteria (U.S. Pat. Nos. 4,657,849 and 4,762,824).
It is also well recognized that oligosaccharides have an influence on the protein or lipid to which they are conjugated (Rademacher et al., Ann. Rev., Biochem., (1988), 57:785). Specific oligosaccharides have been shown to influence proteins, stability, rate of proteolysis, rate of in vivo clearance from the bloodstream, thermal stability and solubility. Changes in the oligosaccharide portion of cell surface carbohydrates have been noted in cells which have become cancerous. Other oligosaccharide changes have been detected during cell differentiation (Toone et al., Tetrahedron Report (1989) 45(17):5365-5422). As such, the significance of oligosaccharides to biological function cannot be understated.
O-glycosidically linked (mucin type) oligosaccharides have been reported on a number of different types of glycoproteins (Sadler, (1984) Biology of Carbohydrates, (Ginsburg and Robbins, eds.) pp. 199-213, Vol. 2, John Wiley and Sons, New York). These structures have been assigned a diverse array of functions, ranging from quite specific such as being involved in cell-cell recognition and host-pathogen interaction, to more general such as providing protection from proteolytic degradation or supplying the appropriate charge and water binding properties to mucous secretions (Sadler (1984) Biology of Carbohydrates (supra); Paulson (1989) Trends Biochem. Sci., 14:272-275; and Jentoft (1990) Trends Biochem. Sci., 15:291-294).
The initial reaction in O-linked oligosaccharide biosynthesis is the transfer of an N-acetylgalactosamine residue from the nucleotide sugar UDP-N-acetylgalactosamine to a serine or threonine residue on the protein acceptor. This reaction, which can occur post-translationally, is catalyzed by UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (hereinafter referred to as GalNAc-transferase or GalNAcT) an intracellular membrane bound enzyme believed to be localized in the secretory pathway.
The exact location(s) of GalNAc-transferase is still controversial. It has been reported that the initial addition of N-acetylgalactosamine to the acceptor protein can take place early (even co-translationally) in the rough endoplasmic reticulum (ER). Other authors have suggested that this reaction is a post-translational event occurring in later ER compartments and/or in the cis region of the Golgi complex (e.g. Hanover et al. (1982) J. Biol. Chem. 257:10172-10177; Roth (1984) J. Cell Biol. 98:399-406; Elhammer and Kornfeld (1984) J. Cell Biol. 98:327-331; Tooze et al. (1988) J. Cell Biol. 106:1475-1487; Deschuyteneer et al. (1988) J. Biol. Chem. 263:2452-2459; Ulmer and Palade (1989) Proc. Natl. Acad. Sci. (U.S.A.) 89:663-667; Wertz et al. (1989) J. Virol. 63:4767-4776; Piller et al. (1989) Eur. J. Biochem. 183:123-135; Piller et al. (1990) J. Biol. Chem. 265:9264-9271. Finally, evidence has also been presented for a model in which transfer of N-acetylgalactosamine to Ser/Thr may occur in several compartments in the secretory pathway, including compartments later than the Golgi complex (Schachter and Brockhausen (1992) Glycoconjugates, Allen and Kisailus, eds., pp. 263-332, Marcel Dekker Inc., New York). Elongation and termination of O-linked oligosaccharides is accomplished by sequential addition of individual monosaccharides by specific transferases (Roseman (1970) Chem. Phys. Lipids 5:270-280); current data suggest that these reactions are localized primarily in the Golgi apparatus (Schachter and Brockhausen, supra).
The fundamental role of oligosaccharides, particularly, O-glycosidically linked (mucin type) oligosaccharides, to biological function in molecular biology has made them the object of considerable research, in particular, considerable efforts have been made in organic synthesis to synthesize these materials. Although synthetic approaches to making carbohydrates are quite developed, this technique suffers notable difficulties which relate to the selective protection and deprotection steps required in the available synthetic pathways. These difficulties, combined with difficulties associated with isolating and purifying carbohydrates, and determining their structures, has made it essentially impossible for synthetic organic chemistry to economically produce valuable carbohydrates.
Enzyme-mediated catalytic synthesis would offer dramatic advantages over the classical synthetic organic pathways, producing very high yields of carbohydrates (e.g., oligosaccharides and/or polysaccharides) economically, under mild conditions in aqueous solutions, and without generating notable amounts of undesired side products. To date, such enzymes, which include glycosyltransferase, are however difficult to isolate, especially from eukaryotic, e.g., mammalian sources, because these proteins are only found in low concentrations, and tend to be membrane-bound. In addition to being difficult to isolate, the acceptor (peptide) specificity of GalNAc-transferase is poorly understood.
It has been reported that in at least three different proteins the acceptor sites glycosylated by the N-acetylglucosaminyltransferase have a common feature. This feature, which appears to lead to nuclear and cytoplasmic O-GlcNAc structures, is an acidic amino acid followed by serine, proline, and then a run of serines and threonines (Haltiwanger et al., 1990). A more narrowly defined acceptor site has been reported for the proteoglycan xylosyltransferase: the acceptor site for this enzyme consists of acidic amino acids closely followed by the tetrapeptide Ser-Gly-Xaa-Gly, where Xaa may be any amino acid (Bourdon et al. 1987). In spite of attempts to define it either by studying the amino acid sequences surrounding glycosylated serine and threonine residues of known location (Hagopian et al., 1971; Hill et al., 1977; Gooley et al., 1991) or by performing in vitro studies on synthetic peptides (Young et al., 1979; Briand et al., 1981; Hughes et al., 1988; Wang et al., 1992), these studies have yielded little conclusive information. In light of the abovenoted considerable value of carbohydrates, there is accordingly a strongly felt need for an improved method for isolation of glycosyltransferase enzyme as well as for studies of the acceptor (peptide) specificity of the enzyme to facilitate its use in carbohydrate synthesis.